Drugs are generally not selective for their target organs or cells and, as a result, they exert toxic side effects. The concept of coupling a therapeutic agent to a carrier molecule (e.g. antibody) with specificity for a defined target cell population is, therefore, an attractive one to achieve site-specific drug delivery. In recent years, for example, a variety of monoclonal antibodies that recognize tumor associated cell-surface antigens have been used as carriers of many of the clinically used anticancer agents, as reviewed in Vogel, C. -W., 1987. Specific and potent cytotoxic activities of such immunoconjugates have been described by investigators in a variety of in vitro systems. Less impressive and even disappointing results were obtained when such immunoconjugates were studied for their immunotherapeutic activity in tumor-bearing animals as well as in cancer patients. These data demonstrate that the concept of immunoconjugates poses a number of difficulties yet to be overcome as it has not been possible to deliver sufficient quantities of drugs coupled to monoclonal antibodies (for review see Vogel., C. -W. 1987).
Early on it was recognized that enzymes may be superior to drugs. Ribosomal inactivating proteins (RIP's) are enzymes and their cytotoxic effect is potentisted by this enzymatic activity. However, like drugs RIP's must be internalized, liberated from the antibody carrier and reach the appropriate intracellular compartment to exert their activity. Moreover, RIP's require antigen expression by every target cell to be effective and they have an inherent cytotoxicity that limits the amount that can be given.
Another enzymatic approach uses a surface active enzyme coupled to an antibody. This antibody-enzyme conjugate would not require internalization and could exert cytotoxic activity on cells in the area. One example of a surface active enzyme that has been used as an antibody conjugate is phospholipase-C, which attacks the phospholipids of all cell membranes directly without requiring internalization. However, the ability of phospholipase to attack the phospholipids in all cells expresses an inherent cytotoxicity, which limits its usefulness (Flickinger and Trost, 1976). Another surface active enzyme used as an antibody conjugate is cobra venom factor (CVF), a complement activating enzyme, which, in addition to not needing to be internalized by the cells, is not inherently cytotoxic (Vogel, C. -W., 1987). However, like all foreign proteins, CVF is highly immunogenic and its use for treating tumor cells is, therefore, limited.
A further approach used antibody-alkaline phosphatase conjugates for dephosphorylation of an etoposide derivative. The problems with this approach are competition with circulating endogenous substrates limiting the enzymatic activity at the target and a high level of enzyme activity in circulation and in non-target tissues, resulting in an increase in unwanted cytotoxic activity (Senter et al, 1988).
A different enzymatic concept used carboxypeptidase-G.sub.2, which cleaves an essential growth factor, folate (Searle et al, 1986). However, a sufficient decrease in folate level requires a higher level of enzyme conjugate in the target tissue than can be achieved using an antibody-enzyme conjugate in-vivo.
In addition to the particular disadvantages as described above, all enzymatic approaches share the problem of immunogenicity. The development of antibodies against the enzymes results in inactivation of the activity by blockage and steric hindrance and rapid clearance of the anti-bodyenzyme from the circulation. For this reason the system suggested by Bagshawe, K. D. (1987) using an enzyme-antibody conjugate for drug activation would, at best, provide useful results in only a single administration, and the immune response may prevent efficacy even then. Bagshowe's concept uses non-mammalian enzymes that do not have human analogues to avoid activation by enzymes other than on the conjugate. However, these enzymes would be highly immunogenic, particularly when conjugated.